Nonidentical subunits of p21H-ras farnesyltransferase. Peptide binding and farnesyl pyrophosphate carrier functions

The protein farnesyltransferase purified from rat brain contains two nonidentical subunits, alpha and beta. The holoenzyme forms a stable complex with [3H]farnesyl pyrophosphate (FPP) that can be isolated by gel filtration. The [3H]FPP is not covalently bound to the enzyme; it is released unaltered when the enzyme is denatured. When incubated with an acceptor such as p21H-ras, the complex transfers [3H]farnesyl from the bound [3H]FPP to the ras protein. This transfer is not sensitive to dilution by unbound FPP, suggesting that the [3H]FPP is bound at a site that leads to direct transfer to the p21H-ras acceptor. Cross-linking studies show that the p21H-ras binds to the lower molecular weight subunit (beta-subunit), raising the possibility that the [3H]FPP binds to the alpha-subunit. If this suggestion can be confirmed, it would invoke a reaction mechanism in which the alpha-subunit acts as a prenyl pyrophosphate carrier that delivers FPP to p21H-ras which is bound to the beta-subunit.     

Synthesis of imidazole-containing analogues of farnesyl pyrophosphate and evaluation of their biological activity on protein farnesyltransferase

With the aim of creating new bisubstrate inhibitors of protein farnesyltransferase (FTase), new carboxylic farnesyl pyrophosphate analogues have been designed and synthesized. The original structures are built around three elements: a prenyl moiety, a 1,4-diacid motif and an imidazole ring. All the compounds were evaluated for their ability to inhibit FTase and compared with the corresponding derivatives lacking the imidazole ring, synthesized for that purpose. These new compounds are not bisubstrate inhibitors probably because the imidazole ring is not in the right position to interact with the zinc atom. However these derivatives display FPP competitive inhibition with a good activity in the carboxylic farnesyl pyrophosphate analogues series.     

A novel prenyltransferase for a small GTP-binding protein having a C-terminal Cys-Ala-Cys structure

smg p25A/rab3A p25 is a member of the small GTP-binding protein superfamily which is implicated in intracellular vesicle transport. smg p25A has a cDNA-predicted C-terminal structure of Cys-Ala-Cys. The protein purified from bovine brain membranes is geranylgeranylated at both the two cysteine residues and carboxyl-methylated at the C-terminal cysteine residue. Two types of prenyltransferase for small GTP-binding proteins have thus far been reported: ras p21 farnesyltransferase (ras p21 FT) and rhoA p21 geranylgeranyltransferase (rhoA p21 GGT). Neither of them geranylgeranylated smg p25A having a C-terminal Cys-Ala-Cys structure. In this paper, a smg p25A GGT was partially purified from bovine brain cytosol and separated from the ras p21 FT and rhoA p21 GGT by column chromatographies. smg p25A GGT transferred the geranylgeranyl moiety from geranylgeranyl pyrophosphate to both the two cysteine residues in the C-terminal Cys-Ala-Cys structure of smg p25A. smg p25A GGT did not use farnesyl pyrophosphate as a substrate and was also inactive on c-Ha-ras p21 and rhoA p21 with either farnesyl pyrophosphate or geranylgeranyl pyrophosphate as a substrate. These results indicate that there are at least three types of prenyltransferase for small GTP-binding proteins in mammalian tissues.     

Some features of methylpyrophosphate hydrolysis by yeast inorganic pyrophosphatase

The interaction of yeast inorganic pyrophosphatase with methylpyrophosphate was studied. In the presence of Mg2+ the rate of hydrolysis of the methylpyrophosphate-Zn2+ complex by the enzyme was shown to decrease. This was accompanied by competition of Zn2+ and Mg2+ for one site of Me2+ binding on the enzyme. The kinetics of combined hydrolysis of zinc methylpyrophosphate and zinc pyrophosphate were studied. It was found that both substrates are hydrolyzed at the same active site of the enzyme. Free methylpyrophosphate when bound to a specific phosphorylation site on the enzyme surface accelerated magnesium pyrophosphate hydrolysis. Some kinetic parameters of this hydrolysis were determined.     

Mammalian protein geranylgeranyltransferase. Subunit composition and metal requirements.

An enzyme capable of specifically modifying, with a geranylgeranyl isoprenoid, candidate proteins containing a consensus prenylation sequence ending in leucine has been purified from bovine brain. This protein geranylgeranyltransferase (PGGT), isolated using affinity chromatography on an immobilized peptide column, contains two subunits with molecular masses of 48 and 43 kDa, designated alpha and beta, respectively. An antiserum raised to the alpha subunit of the related enzyme, protein farnesyltransferase (PFT), also recognizes this chromatographically identical alpha-subunit of the PGGT by immunoblot analysis. The PGGT and PFT enzymes from bovine brain are shown to be dependent on both Mg2+ and Zn2+ for optimal activity. Demonstration of the Zn2+ dependence of the enzymes requires prolonged incubation or purification in the presence of a chelating agent; we therefore propose that these enzymes be placed into the category of metalloenzymes. Under optimal assay conditions, these enzymes show high specificity toward their prenyl diphosphate substrates, with only a weak competition observed with farnesyl diphosphate in the PGGT reaction or geranylgeranyl diphosphate in the PFT reaction. The two enzymes are differentially sensitive to several detergents tested to determine suitable ones for product stabilization in the reactions. These results confirm previous predictions on the subunit structure of the PGGT and provide an avenue to initiating a molecular analysis of the geranylgeranyl modification of many mammalian proteins.     

Substrate Binding of avian liver prenyltransferase.

Prenyltransferase (farnesyl pyrophosphate synthetase) was purified from avian liver and characterized by Sephadex and sodium dodecyl sulfate gel chromatography, peptide mapping, and end-group analysis. The enzyme is 85 800 +/- 4280 daltons and consists of two identical subunits as judged by sodium dodecyl sulfate gel electrophoresis, peptide mapping, and end-group analysis. Chemical analysis of the protein revealed no lipid or carbohydrate components. Avian prenyltransferase synthesizes farnesyl pyrophosphate from either dimethylallyl or geranyl pyrophosphate and isopentenyl pyrophosphate. A lower rate of geranylgeranyl pyrophosphate synthesis from farnesyl pyrophosphate and isopentenyl pyrophosphate was also demonstrated. Michaelis constants for farnesyl pyrophosphate synthesis are 0.5 muM for both isopentenyl pyrophosphate and geranyl pyrophosphate. The V max for the reaction is 1990 nmol min-1 mg-1 (170 mol min-1 mol-1 enzyme). Substrate inhibition by isopentenyl pyrophosphate is evident at high isopentenyl pyrophosphate and low geranyl pyrophosphate concentrations. Michaelis constants for geranylgeranyl pyrophosphate synthesis are 9 muM for farnesyl pyrophosphate and 20 muM for isopentenyl pyrophosphate. The Vmax is 16 nmol min-1 mg-1 (1.4 mol min-1 mol-1 enzyme). Two moles of each of the allylic substrates is bound per mol of enzyme. The apparent dissociation constants for dimethylallyl, geranyl, and farnesyl pyrophosphates are 1.8, 0.17, and 0.73 muM, respectively. Dimethylallyl and geranyl pyrophosphates bound competitively to prenyltransferase with one-for-one displacement. Four moles of isopentenyl pyrophosphate was bound per mole of enzyme. Citronellyl pyrophosphate, an analogue of geranyl pyrophosphate, was competitive with the binding of 2 of the 4 mol of isopentenyl pyrophosphate bound. The data are interpreted to indicate that each subunit of avian liver prenyltransferase has a single allylic binding site accommodating dimethylallyl, geranyl, and farnesyl pyrophosphates, and one binding site for isopentenyl pyrophosphate. In the absence of an allylic pyrophosphate or analogue, isopentenyl pyrophosphate also can bind to the allylic site.     

Heptaprenyl pyrophosphate synthetase from Bacillus subtilis

Heptaprenyl pyrophosphate synthetase was detected in partially purified extracts of Bacillus subtilis. The enzyme catalyzed the synthesis of all-trans C35 prenyl pyrophosphate from isopentenyl pyrophosphate and farnesyl or geranylgeranyl pyrophosphate, but it did not catalyze a reaction between isopentenyl pyrophosphate and either dimethylallyl or geranyl pyrophosphate. The enzyme reaction proceeded with an elimination of 2-pro-R hydrogen of isopentenyl pyrophosphate without accumulation of any prenyl pyrophosphate shorter than C35. The molecular weight of the enzyme was estimated by gel filtration to be 45,000. Michaelis constants for isopentenyl, farnesyl, and geranylgeranyl pyrophosphate were 12.8, 13.3, and 8.3 microM, respectively.     

Crystal structures of undecaprenyl pyrophosphate synthase in complex with magnesium, isopentenyl pyrophosphate, and farnesyl thiopyrophosphate: roles of the metal ion and conserved residues in catalysis

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes the consecutive condensation reactions of a farnesyl pyrophosphate (FPP) with eight isopentenyl pyrophosphates (IPP), in which new cis-double bonds are formed, to generate undecaprenyl pyrophosphate that serves as a lipid carrier for peptidoglycan synthesis of bacterial cell wall. The structures of Escherichia coli UPPs were determined previously in an orthorhombic crystal form as an apoenzyme, in complex with Mg(2+)/sulfate/Triton, and with bound FPP. In a further search of its catalytic mechanism, the wild-type UPPs and the D26A mutant are crystallized in a new trigonal unit cell with Mg(2+)/IPP/farnesyl thiopyrophosphate (an FPP analogue) bound to the active site. In the wild-type enzyme, Mg(2+) is coordinated by the pyrophosphate of farnesyl thiopyrophosphate, the carboxylate of Asp(26), and three water molecules. In the mutant enzyme, it is bound to the pyrophosphate of IPP. The [Mg(2+)] dependence of the catalytic rate by UPPs shows that the activity is maximal at [Mg(2+)] = 1 mm but drops significantly when Mg(2+) ions are in excess (50 mm). Without Mg(2+), IPP binds to UPPs only at high concentration. Mutation of Asp(26) to other charged amino acids results in significant decrease of the UPPs activity. The role of Asp(26) is probably to assist the migration of Mg(2+) from IPP to FPP and thus initiate the condensation reaction by ionization of the pyrophosphate group from FPP. Other conserved residues, including His(43), Ser(71), Asn(74), and Arg(77), may serve as general acid/base and pyrophosphate carrier. Our results here improve the understanding of the UPPs enzyme reaction significantly.     

Structure and mechanism of the farnesyl diphosphate synthase from Trypanosoma cruzi: implications for drug design

Typanosoma cruzi, the causative agent of Chagas disease, has recently been shown to be sensitive to the action of the bisphosphonates currently used in bone resorption therapy. These compounds target the mevalonate pathway by inhibiting farnesyl diphosphate synthase (farnesyl pyrophosphate synthase, FPPS), the enzyme that condenses the diphosphates of C5 alcohols (isopentenyl and dimethylallyl) to form C10 and C15 diphosphates (geranyl and farnesyl). The structures of the T. cruzi FPPS (TcFPPS) alone and in two complexes with substrates and inhibitors reveal that following binding of the two substrates and three Mg2+ ions, the enzyme undergoes a conformational change consisting of a hinge-like closure of the binding site. In this conformation, it would be possible for the enzyme to bind a bisphosphonate inhibitor that spans the sites usually occupied by dimethylallyl diphosphate (DMAPP) and the homoallyl moiety of isopentenyl diphosphate. This observation may lead to the design of new, more potent anti-trypanosomal bisphosphonates, because existing FPPS inhibitors occupy only the DMAPP site. In addition, the structures provide an important mechanistic insight: after its formation, geranyl diphosphate can swing without leaving the enzyme, from the product site to the substrate site to participate in the synthesis of farnesyl diphosphate.     

Biosynthesis of the Macrocyclic Diterpene Casbene in Castor Bean (Ricinus communis L.) Seedlings : The Purification and Properties of Farnesyl Transferase from Elicited Seedlings

Farnesyl transferase (farnesyl pyrophosphate: isopentenyl pyrophosphate farnesyl transferase; geranylgeranyl pyrophosphate synthetase) was purified at least 400-fold from extracts of castor bean (Ricinus communis L.) seedlings that were elicited by exposure for 10 h to Rhizopus stolonifer spores. The purified enzyme was free of isopentenyl pyrophosphate isomerase and phosphatase activities which interfere with prenyl transferase assays. The purified enzyme showed a broad optimum for farnesyl transfer between pH 8 and 9. The molecular weight of the enzyme was estimated to be 72,000 ± 3,000 from its behavior on a calibrated G-100 Sephadex molecular sieving column. Mg²⁺ ion at 4 millimolar gave the greatest stimulation of activity; Mn²⁺ ion gave a small stimulation at 0.5 millimolar, but was inhibitory at higher concentrations. Farnesyl pyrophosphate (K_m = 0.5 micromolar) in combination with isopentenyl pyrophosphate (K_m = 3.5 micromolar) was the most effective substrate for the production of geranylgeranyl pyrophosphate. Geranyl pyrophosphate (K_m = 24 micromolar) could replace farnesyl pyrophosphate as the allylic pyrophosphate substrate, but dimethylallyl pyrophosphate was not utilized by the enzyme. One peak of farnesyl transferase activity (geranylgeranyl pyrophosphate synthetase) and two peaks of geranyl transferase activity (farnesyl pyrophosphate synthetases) from extracts of whole elicited seedlings were resolved by DEAE A-25 Sephadex sievorptive ion exchange chromatography. These results suggest that the pathway for geranylgeranyl pyrophosphate synthesis in elicited castor bean seedlings involves the successive actions of two enzymes—a geranyl transferase which utilizes dimethylallypyrophosphate and isopentenyl pyrophosphate as substrates and a farnesyl transferase which utilizes the farnesyl pyrophosphate produced in the first step and isopentenyl pyrophosphate as substrates.     

Role of metals in the reaction catalyzed by protein farnesyltransferase

Protein farnesyltransferase catalyzes the posttranslational farnesylation of several proteins involved in signal transduction, including Ras, and is a target enzyme for antitumor therapies. Efficient product formation catalyzed by protein farnesyltransferase requires an enzyme-bound zinc cation and high concentrations of magnesium ions. In this work, we have measured the pH dependence of the chemical step of product formation, determined under single-turnover conditions, and have demonstrated that the prenylation rate constant is enhanced by two deprotonations. Substitution of the active site zinc by cadmium demonstrated that one of the ionizations reflects deprotonation of the metal-coordinated thiol of the peptide "CaaX" motif, pK(a1) = 6.0. These data provide additional evidence for the direct involvement of a metal-coordinated sulfur nucleophile in catalysis. The second ionization was assigned to a hydroxyl on the pyrophosphate moiety of farnesyl pyrophosphate, pK(a2) = 7.4. Deprotonation of this group is important for binding of magnesium. This second ionization is not observed for catalysis in the absence of magnesium or when the substrate is farnesyl monophosphate. These data indicate that the maximal rate constant for prenylation requires formation of a zinc-coordinated thiolate nucleophile and enhancement of the electrophilic character at C1 of the farnesyl chain by magnesium ion coordination of the pyrophosphate leaving group.     

Farnesyl pyrophosphate synthetase from Bacillus subtilis

Farnesyl pyrophosphate synthetase was detected in extracts of Bacillus subtilis and partially purified by Sephadex G-100, hydroxylapatite, and DEAE-Sephadex chromatography. The enzyme catalyzed the exclusive formation of all-trans farnesyl pyrophosphate from isopentenyl pyrophosphate and either dimethylallyl or geranyl pyrophosphate. Mg2+ was essential for the catalytic activity and Mn2+ was less effective. The enzyme was slightly activated by sulfhydryl reagents. This enzyme was markedly stimulated by K+, NH4+, or detergents such as Triton X-100 and Tween 80, unlike the known farnesyl pyrophosphate synthetases from eucaryotes. The molecular weight of the enzyme was estimated by gel filtration to be 67,000. The Michaelis constants for dimethylallyl and geranyl pyrophosphate were 50 microM and 18 microM, respectively.     

Nonfarnesylated tetrapeptide inhibitors of protein farnesyltransferase

The protein farnesyltransferase from rat brain was previously shown to be inhibited competitively by tetrapeptides that conform to the consensus Cys-A1-A2-X, where A1 and A2 are aliphatic amino acids and X is methionine, serine, or phenylalanine. In the current studies we use a thin layer chromatography assay to show that most of these tetrapeptides are themselves farnesylated by the purified enzyme. Two classes of tetrapeptides are not farnesylated and therefore act as true inhibitors: 1) those that contain an aromatic residue at the A2 position and 2) those that contain penicillamine (beta,beta-dimethylcysteine) in place of cysteine. The most potent of these pure inhibitors was Cys-Val-Phe-Met, which inhibited farnesyltransferase activity by 50% at less than 0.1 microM. These data indicate that the inclusion of bulky aromatic or methyl residues in a tetrapeptide can abolish prenyl group transfer without blocking binding to the enzyme. This information should be useful in the design of peptides or peptidomimetics that inhibit farnesylation and thus block the action of p21ras proteins in animal cells.     

The synthesis and biological evaluation of a series of potent dual inhibitors of farnesyl and geranyl-Geranyl protein transferases

We have prepared a series of potent, dual inhibitors of the prenyl transferases farnesyl protein transferase (FPTase) and geranyl-geranyl protein transferase I (GGPTase). The compounds were shown to possess potent activity against both enzymes in cell culture. Mechanistic analysis has shown that the compounds are CAAX competitive for FPTase inhibition but geranyl-geranyl pyrophosphate (GGPP) competitive for GGPTase inhibiton.     

Is pyrophosphate an analog of adenosine diphosphate for beef heart mitochondrial F1-ATPase

Beef heart mitochondrial F1 possesses three pyrophosphate-binding sites, which comprises one high affinity binding site (Kd approximately equal to 1 microM) and two lower affinity sites (Kd approximately equal to 20 microM). High affinity pyrophosphate binding required the presence of Mg2+ in the incubation medium. Pyrophosphate competed with ADP, but not with Pi for binding to mitochondrial F1. Upon binding of 3 mol of pyrophosphate/mol of F1, one of the three tightly bound nucleotides present in native F1 was released. Like ADP and in contrast to Pi, pyrophosphate enhanced the fluorescence intensity of F1-bound aurovertin, and it prevented the photolabeling of F1 by 2-azido-ADP. As aurovertin and 2-azido-ADP are ligands of the beta subunit of F1, it is likely that pyrophosphate binds preferentially to the beta subunit. Whereas the binding affinity of F1 for Pi was increased by concentrations of pyrophosphate lower than 100 microM, it was decreased by a higher concentration of pyrophosphate. This biphasic effect of pyrophosphate on Pi binding was not observed with ADP, which, at all concentrations tested, inhibited Pi binding. Except for the effect of pyrophosphate on Pi binding to F1, for all the other effects, pyrophosphate mimicked ADP. It is suggested that pyrophosphate and ADP share the same binding site on F1 and that pyrophosphate interacts with the same amino acid residues as those interacting with the alpha and beta phosphate groups of ADP.     

Inhibition of farnesyl transferases from malignant and non-malignant cultured human lymphocytes by prenyl substrate analogues.

Cytosolic prenyl transferases from two human lymphoid tissue-derived cell lines, IM-9 and Molt-4 cells, are shown to isoprenylate recombinant p21H-ras. Isoprenylation was inhibited by an N-acetylated pentapeptide (N-Ac-Lys-Cys-Val-Leu-Ser), c,t-farnesyl diphosphate, c,t,t-geranylgeranyl diphosphate, t,t,t-geranylgeranyl diphosphate and a photolabile farnesyl diphosphate analogue. c,t-Farnesyl and t,t,t-geranylgeranyl monophosphates were also effective inhibitors of the Molt-4 enzyme but not the IM-9 enzyme.     

Towards the synthesis of bisubstrate inhibitors of protein farnesyltransferase: Synthesis and biological evaluation of new farnesylpyrophosphate analogues

Protein farnesyltransferase (FTase) has recently appeared as a new target of parasitic diseases, a field poor in drugs in development. With the aim of creating new bisubstrate inhibitors of FTase, new farnesyl pyrophosphate analogues have been studied. Farnesyl analogues with a malonic acid function exhibited the best inhibitory activity on FTase. This group was introduced into our imidazole-containing model leading to new compounds with submicromolar activities. Kinetic experiments have been realized to determine their binding mode to the enzyme.     

Inhibition of purified p21ras farnesyl:protein transferase by Cys-AAX tetrapeptides

We report the identification, purification, and characterization of a farnesyl:protein transferase that transfers the farnesyl moiety from farnesyl pyrophosphate to a cysteine in p21ras proteins. The enzyme was purified approximately 60,000-fold from rat brain cytosol through use of a chromatography step based on the enzyme's ability to bind to a hexapeptide containing the consensus sequence (Cys-AAX) for farnesylation. The purified enzyme migrated on gel filtration chromatography with an apparent molecular weight of 70,000-100,000. High resolution SDS-polyacrylamide gels showed two closely spaced approximately 50 kd protein bands in the final preparation. The enzyme was inhibited competitively by peptides as short as 4 residues that contained the Cys-AAX motif. These peptides acted as alternative substrates that competed with p21H-ras for farnesylation. Effective peptides included the COOH-terminal sequences of all known p21ras proteins as well as those of lamin A and B.     

Finding a needle in the haystack: computational modeling of Mg2+ binding in the active site of protein farnesyltransferase

Studies aimed at elucidating the unknown Mg2+ binding site in protein farnesyltransferase (FTase) are reported. FTase catalyzes the transfer of a farnesyl group to a conserved cysteine residue (Cys1p) on a target protein, an important step for proteins in the signal transduction pathways (e.g., Ras). Mg2+ ions accelerate the protein farnesylation reaction by up to 700-fold. The exact function of Mg2+ in catalysis and the structural characteristics of its binding remain unresolved to date. Molecular dynamics (MD) simulations addressing the role of magnesium ions in FTase are presented, and relevant octahedral binding motifs for Mg2+ in wild-type (WT) FTase and the Dβ352A mutant are explored. Our simulations suggest that the addition of Mg2+ ions causes a conformational change to occur in the FTase active site, breaking interactions known to keep FPP in its inactive conformation. Two relevant Mg2+ ion binding motifs were determined in WT FTase. In the first binding motif, WT1, the Mg2+ ion is coordinated to D352β, zinc-bound D297β, two water molecules, and one oxygen atom from the α- and β-phosphates of farnesyl diphosphate (FPP). The second binding motif, WT2, is identical with the exception of the zinc-bound D297β being replaced by a water molecule in the Mg2+ coordination complex. In the Dβ352A mutant Mg2+ binding motif, D297β, three water molecules, and one oxygen atom from the α- and β-phosphates of FPP complete the octahedral coordination sphere of Mg2+. Simulations of WT FTase, in which Mg2+ was replaced by water in the active site, recreated the salt bridges and hydrogen-bonding patterns around FPP, validating these simulations. In all Mg2+ binding motifs, a key hydrogen bond was identified between a magnesium-bound water and Cys1p, bridging the two metallic binding sites and, thereby, reducing the equilibrium distance between the reacting atoms of FPP Cys1p. The free energy profiles calculated for these systems provide a qualitative understanding of experimental results. They demonstrate that the two reactive atoms approach each other more readily in the presence of Mg2+ in WT FTase and mutant. The flexible WT2 model was found to possess the lowest barrier toward the conformational change, suggesting it is the preferred Mg2+ binding motif in WT FTase. In the mutant, the absence of D352β makes the transition toward a conformational change harder. Our calculations find support for the proposal that D352β performs a critical role in Mg2+ binding and Mg2+ plays an important role in the conformational transition step.     

Squalene synthetase. Solubilization and partial purification of squalene synthetase, copurification of presqualene pyrophosphate and squalene synthetase activities

Squalene synthetase (farnesyldiphosphate:farnesyldiphosphate farnesyltransferase, EC 2.5.1.21) is an intrinsic microsomal protein that catalyzes the synthesis of squalene from farnesyl pyrophosphate via the intermediate presqualene pyrophosphate. We have solubilized this enzyme from yeast with a mixture of the detergents N-octyl beta-D-glucopyranoside and Lubrol PX. Approximately 50-fold purification of the solubilized activities has been achieved by chromatography on DEAE-cellulose and hydroxylapatite and by isoelectric focusing. The most highly purified preparation has one major band of protein with a molecular weight of 53,000 as estimated by electrophoresis under denaturing conditions. The enzyme may also have been modified by proteolysis during isolation since a 47,000 molecular weight species was also found. The two activities, presqualene pyrophosphate synthetase and squalene synthetase, copurified during isolation.